Insulators are conserved genomic elements that organize chromosomes into independent functional domains to maintain transcriptional fidelity. These elements protect gene expression from positive and negative regulatory effects by blocking interactions between enhancers, silencers, and promoters. The goal of our studies is to understand molecular mechanisms used by insulators to organize eukaryotic genomes into transcriptional domains. In studies described herein, the Drosophila gypsy insulator will be used as model to elucidate mechanisms of insulator action. The gypsy insulator is a well-studied insulator that was isolated from the gypsy retrovirus. This insulator is the only one known that requires a single DNA binding protein, the Suppressor of Hairy-wing [Su(Hw)]. Once bound to chromosomes, the Su(Hw) protein recruits the BTB/POZ domain protein, Modifier67.2, to generate a protein complex that establishes insulator activity. Mutations in the genes encoding the gypsy insulator proteins are available, making the system amenable to genetic manipulation. The gypsy insulator proteins associate with hundreds of genomic sites that are not sites of gypsy retroviral insertion. These observation suggest that Su(Hw) and Modifier67.2 have global roles in establishing independent regulatory domains in the Drosophila genome. We will test this hypothesis by determining the DNA and proteins requirements of endogenous Su(Hw) sites and define their role in organization of regulatory domains throughout chromosomes. These data will provide insights into mechanisms used to delimit transcriptional regulatory domains within eukaryotic genomes, thereby providing insights into fundamental questions of control of enhancer and silencer function. Understanding mechanisms of insulators action has therapeutic utility for gene therapy, as inclusion of insulators within gene transfer vectors can improve the expression of transgenes, while preventing inappropriate regulatory effects of gene transfer vectors on endogenous gene expression.